Federico E. Turkheimer, Paul Edison, Nicola Pavese, Federico Roncaroli, Alexander Hammers, Alex Anderson, Alexander Gerhard, Rainer Hinz, Yen F. Tai, David J. Brooks PET Modelling Group, CSC-MRC, Hammersmith Hospital, London, UK Division of Neuroscience, Imperial College London, UK Hammersmith-Imanet, Hammersmith Hospital, London, UK © Imperial College London Supervised reference region extraction for the quantification of [11C]-(R)-PK11195 brain studies.

© Imperial College London TALK IN A NUTSHELL The Peripheral Benzodiazepine Receptor Existing Methodology for [ 11 C]-(R)-PK11195 Aims of the work Quantitative Assessment of New Methodology Conclusions

© Imperial College London The Peripheral Benzodiazepine Receptor (PBR) The PBR is a nuclear encoded mitochondrial protein. The PBR is abundant in peripheral organs (particularly adrenal glands and kidney) and haematogenous cells. The function of the PBR still needs full elucidation but the receptor plays an important role in steroid synthesis and in the regulation of immunological responses in mononuclear phagocytes

© Imperial College London The PBR in Diseases of the CNS High PBR has been observed in infiltrating blood- borne cells and activated microglia. Significant microglial activation occurs after mild to severe neuronal damage resulting from traumatic, inflammatory, degenerative and neoplastic disease. Microglia are activated in the surroundings of focal lesions but also in the distant, anterograde and retrograde projection areas of the lesioned neural pathway.

© Imperial College London PET Imaging with [ 11 C]-(R)- PK11195 PK11195 is a selective ligand for the peripheral benzodiazepine receptor. PET imaging using the molecular marker [11C]-(R)- PK11195 now provides an indicator of active disease in the brain. Wide applicability including multiple sclerosis, amyotrophic lateral sclerosis, dementia, Parkinson’s disease, corticobasal degeneration, Huntington’s disease, epilepsy, schizophrenia, gliomas etc.

© Imperial College London The Peripheral Benzodiazepine Receptor Existing Methodology for [ 11 C]-(R)-PK11195 Aims of the work Quantitative Assessment of New Methodology Conclusions TALK IN A NUTSHELL

© Imperial College London Modelling [ 11 C]-(R)-PK11195 So far, quantification adopted some form of normalization to a reference region or application of the simplified reference tissue model (SRTM) (BP ref in gray matter ~[ ]). Plasma kinetic model (2 compartments, 4 rate constants) recently developed (Kropholler et al., 2005). BP pl (gray matter) = K 3 /K 4 ~=1.6. Differences between BP pl and BP ref largely due to non-specific binding. However BP pl corrected for non-specific binding still higher than BP ref. Specific binding in the reference?

© Imperial College London Reference Region for [ 11 C]-(R)- PK11195 studies The selection of a reference region devoid of PBR is a challenging task. - Microglia are distributed ubiquitously - Activation may occur along projections in healthy appearing tissue. - Microglia activation associated with aging. Informed choice of a reference region based on post-mortem data occasionally feasible. Unsupervised cluster analysis segments voxels into classes and one selects as reference the class with kinetic behaviour closest to healthy grey matter.

© Imperial College London Project Aims: Characterize the distribution of the PBR in normal brain with immunohistochemistry. Devise an automatic reference region extraction methodology: - Able to select the “right” reference region - Highly reproducible Adjust quantification methodology to accommodate the newly extracted reference

© Imperial College London Immunohistochemistry Monoclonal Antibody (supplied by Dr. Casellas, Sanofi Synthelabo, Montpellier, France) Epitope aa-YHGWHGGRRLPE (2 isoforms at BLAST search) Peripheral-type Benzodiazepine Receptor – [Homo sapiens] – 169 aa (gi ) mappwvpamg ftlapslgcf vgsrfvhgeg lrwyaglqkp swhpphwvlg pvwgtlysamgygsylvwke lggftekavg spgplhwaag pelgmaphll garqmgwalv dlllvsgaaaattvawyqvs plaarllypy lawlafattl nycvwrdnhg whggrrlpe Peripheral-type Benzodiazepine Receptor [Homo sapiens] – 62 aa (gi ) … … alvdlllvsg aaaattvawy qvsplaarll ypylawlafa ttlnycvwrd nhgwhggrrlpe BLAST

© Imperial College London Immunohistochemistry-Results Medium sized artery in cerebellar white matter Small artery in frontal cortex Choroid Plexus Ependyma

© Imperial College London The Peripheral Benzodiazepine Receptor Existing Methodology for [ 11 C]-(R)-PK11195 Aims of the work Distribution of the PBR in the Normal Brain Reference Region Extraction Plasma Input Modelling Test-Retest Reliability Conclusions TALK IN A NUTSHELL

© Imperial College London Supervised Reference Region Algorithm Normalization: each frame was normalized by subtracting its mean and dividing it by its standard deviation to create a unit input (Chen et a., 2000) Healthy Tissue Classes: 12 Normal Controls. - Normal gray and white matter: segmentation of MRI volumes using SPM2 (FIL, Queen Sq, London). - Blood Pool: manual ROIs on vinous sinus. - Muscle:manual ROIs on sterno-mastoid muscle. - Skull: manual ROIs. High Density PBR Class: 3 Huntington’s patients. - Striatum & Globus Pallidus: Manual ROI

© Imperial College London Supervised Reference Region Algorithm

© Imperial College London Segmentation: Results Huntington’s patient: gray matter class (>90%)

© Imperial College London Segmentation: Results(II) Huntington’s patient: high PBR class (>90%)

© Imperial College London Segmentation: Results(III) Huntington’s patient: blood pool class (>90%)

© Imperial College London Segmentation: TACs Huntington’s patient: Time-activity curves for kinetic classes

© Imperial College London The Peripheral Benzodiazepine Receptor Existing Methodology for [ 11 C]-(R)-PK11195 Aims of the work Distribution of the PBR in the Normal Brain Reference Region Extraction Plasma Input Modelling Test-Retest Reliability Conclusions TALK IN A NUTSHELL

© Imperial College London Plasma Input Modelling: Data Six healthy controls injected with 185 MBq of [ 11 C]- (R)-PK11195 PET scanner: ECAT EXACT 3D (CTI/Siemens) - 95 planes, no septa. - Reconstructed with 3D-FBP, resolution 5.1 mm (FWHM) transaxially and 5.9 mm FWHM axially. Acquisition: 60 mins, 18 time-frames. Arterial sampling: - 15mins continuous plus 8 discrete samples. - 5 discrete samples used for metabolite analysis

© Imperial College London Normal Control no. 1 Normal Control no. 2 Plasma Input Modelling: Results Spectral analysis of global gray matter for healthy controls: persistent finding of a slow kinetic component

© Imperial College London Normal Control no. 1 Normal Control no. 2 Plasma Input Modelling: Results (II) Subtraction of the slow kinetic component from healthy gray matter generated TACs close to the reference TACs extracted by the supervised algorithm >>> The slow kinetic component is located in the vasculature (smooth muscles). Similar PK11195 kinetic reported in the heart (Charbonneau et al, 1986)

© Imperial College London Plasma Input Modelling: BP calculation Reference Input Modelling: Plasma Input Modelling: V tg = Volume of distribution – target region V ref = Volume of distribution – extracted reference Volumes of distributions were calculated with Rank- Shaping Spectral Analysis (RS-ESA - Bayesian, no positivity constraints).

© Imperial College London Plasma Input Modelling: Results ROIs: -whole gray matter -whole white matter -cerebellum -thalamus -parietal cortex High correlation (r=0.811, p<10 -5 ). BPs from plasma and reference input in excellent concordance. Meaningful dynamic range (white matter BP ~0, large thalamic BPs). Thalamus White matter

© Imperial College London The Peripheral Benzodiazepine Receptor Existing Methodology for [ 11 C]-(R)-PK11195 Aims of the work Distribution of the PBR in the Normal Brain Reference Region Extraction Plasma Input Modelling Test-Retest Reliability Conclusions TALK IN A NUTSHELL

© Imperial College London Test-retest: Data 4 Alzheimer patients injected with 185 MBq of [ 11 C]PK11195, scanned twice at <6 weeks interval PET scanner: ECAT EXACT 3D (CTI/Siemens) No arterial sampling available. Reference region extracted with: - unsupervised clustering (Gunn et al., 1998) - supervised method BP parametric maps calculated with RPM. BP maps normalized to MNI space using co- registered MRI volumes. Automatic ROI sampling using Maximum Probability Atlas (Hammers et al, 2003).

© Imperial College London Test-retest: Results

© Imperial College London Test-retest: Results (II)

© Imperial College London The new clustering procedure can select a “proper” reference in gray matter filtering out contributions of white matter and specific PBR binding. Tissue kinetics contain a slow specific component of vascular origin. Difference in kinetics due to higher affinity of the PBR isoform (?). Excellent agreement between BPs obtained from plasma and reference input modelling. BPs display meaningful dynamic range. Reference extraction significantly more reliable than previous methodology. Conclusions

© Imperial College London Acknowledgements MRC-CSC-PET Modelling Group: Alex Anderson MRC-CSC-PET Neurology Group: Paul Edison, Nicola Pavese, Alexander Hammers, Yen Tai, David Brooks Neuropathology Dept, Imperial College London: Federico Roncaroli Psychiatry Dept, University of Mainz: Alexander Gerhard Wolfson Molecular Imaging Centre, University of Manchester: Rainer Hinz, Marie-Claude Asselin

© Imperial College London

Immunohistochemistry-Positive Control Adrenal Glands 21 kDa MW Adrenal Glands Brain Western Blot